Nucleic acid amplification techniques have provided powerful tools for detection and analysis of small amounts of nucleic acids. The extreme sensitivity of such methods has lead to attempts to develop them for early diagnosis of infectious and genetic diseases, isolation of genes for analysis, and detection of specific nucleic acids in forensic medicine. Nucleic acid amplification techniques can be grouped according to the temperature requirements of the procedure. The polymerase chain reaction (PCR), ligase chain reaction (LCR) and transcription-based amplification require repeated cycling of the reaction between high and low temperatures to regenerate single stranded target molecules for subsequent cycles of amplification. In contrast, methods such as Strand Displacement Amplification (SDA), self-sustained sequence replication (3SR), Nucleic Acid Sequence Based Amplification (NASBA) and the Q.beta. replicase system are isothermal reactions which can be performed at a constant temperature.
In the PCR, the temperature of the reaction is raised after primer extension to separate the newly-synthesized strand from the template. The temperature is then lowered to reanneal the primers and repeat the extension process. The steps of the PCR reaction therefore occur in discrete phases or cycles as a result of the temperature constraints of the reaction. In contrast, in Strand Displacement Amplification (SDA) and other isothermal amplification reactions, extension of primers, displacement of single stranded extension products, annealing of primers to the extension products (or the original target sequence) and subsequent extension of the primers occur concurrently in the reaction mix. Conventional SDA (performed at lower temperatures, usually about 35-45.degree. C.) is described by G. T. Walker, et al. (1992a. Proc. Natl. Acad. Sci. USA 89, 392-396 and 1992b. Nuc. Acids Res. 20, 1691-1696, U.S. Patents). A thermophilic version of the SDA reaction (tSDA) has recently been developed, and is performed at a higher, but still constant, temperature using thermostable polymerases and restriction endonucleases. tSDA has the advantage of increased specificity and a more rapid reaction time. The reaction is performed essentially as conventional SDA, with substitution of a thermostable polymerase and a thermostable restriction endonuclease. The temperature of the reaction is adjusted to a higher temperature suitable for the selected thermophilic enzymes (typically between about 45.degree. C. and 60.degree. C.) and the conventional restriction endonuclease recognition/cleavage site is replaced by the appropriate restriction endonuclease recognition/cleavage site for the selected thermostable endonuclease. Also in contrast to conventional SDA, the practitioner may include the enzymes in the reaction mixture prior to the initial heat denaturation step if they are sufficiently stable at that temperature.
Targets for amplification by SDA may be prepared by fragmenting larger nucleic acids using the endonuclease used in the SDA reaction. However, when the target is not flanked by the necessary restriction endonuclease recognition sites for fragmentation, target nucleic acids having appropriate restriction endonuclease recognition sites for nicking in the SDA reaction may be generated as described by Walker, et al. (1992b, supra and U.S. Pat. No. 5,270,184). As in SDA, the individual steps of the target generation reaction occur concurrently and continuously, generating targets with the terminal recognition sequences required for nicking by the restriction enzyme in SDA. As all of the components of the SDA reaction are present in the target generation reaction, generated targets automatically and continuously enter the SDA cycle and are amplified.
As the mRNA transcripts of an expressed gene are generally present in the cell in greater copy number than the gene itself, detection of RNA targets alone, or both RNA targets and DNA targets may overcome problems of inadequate sensitivity in some amplification reactions. Amplification of RNA and DNA targets is often desirable for diagnostic application of amplification technologies, as this gives the greatest number of amplifiable targets per sample, and, as a result, the greatest diagnostic sensitivity. Amplification of RNA targets is also useful for diagnostic monitoring of RNA-related conditions such as certain viremias, upregulation of cancer genes, etc. Amplification of RNA targets is referred to as "reverse transcription amplification," the best known method being reverse transcription PCR (rtPCR, J. W. Larrick. 1992. Trends Biotechnology 10, 146-152). rtPCR is often performed in sequential steps, the first being a reaction in which a reverse transcriptase is used to generate a cDNA copy of the RNA target sequence. Reverse transcriptase is then inactivated, and in the second step DNA polymerase is added and the cDNA is amplified in a conventional PCR reaction. This format is consistent with the PCR reaction itself, which occurs in discrete phases of temperature cycling. Recently, rtPCR has been performed using a single polymerase for both reverse transcription and DNA polymerization (EP 0 632 134 A2). It has been reported that thermostable DNA-dependent DNA polymerases such as Taq and Tth have significant reverse transcriptase activity (U.S. Pat. No. 5,322,770) when evaluated under reaction conditions appropriate for reverse transcription. These assays did not require that the DNA-dependent DNA polymerase displace the cDNA from the RNA template or incorporate modified dNTPs using an RNA template.
Reverse transcriptases (typically viral enzymes) are very active in producing cDNA using RNA as a template for replication. AMV reverse transcriptase has also been shown to incorporate thiolated dNTPs into the cDNA (P. A. Bartlett and F. Eckstein. 1982. J Biol. Chem. 257, 8879-8884), although it was not previously known whether other reverse transcriptases had this capability. Strand displacing activity has also been reported for certain reverse transcriptases, but it was not previously known whether these enzymes would strand displace a cDNA containing modified nucleotides from an RNA template, as is required for SDA.
The following terms are defined herein as follows:
An amplification primer is a primer for amplification of a target sequence by hybridization and extension of the primer. For SDA, the 3' end of the amplification primer is a target binding sequence which hybridizes at the 3' end of the target sequence. The amplification primer further comprises a recognition site for a restriction endonuclease 5' to the target binding sequence, generally near its 5' end. The restriction endonuclease recognition site is a nucleotide sequence recognized by a restriction endonuclease which will nick a double stranded recognition site for the restriction endonouclease when the recognition site is hemimodified, as described by Walker, et al. (1992a), supra. A hemimodified recognition site is a double stranded recognition site for a restriction endonuclease in which one strand contains at least one derivatized nucleotide which prevents cutting of one of the strands of the duplex by the restriction endonuclease. "Nicking" refers to this modified activity, in which only one strand of the duplex is cut by the restriction endonuclease, in contrast to typical double-stranded cleavage. Any hemimodified restriction endonuclease recognition site which is nickable by a restriction endonuclease is suitable for use in SDA. Amplification primers for SDA are designated S.sub.1 and S.sub.2 by Walker, et al. (1992b), supra. Alpha-thio modified deoxyribonucleoside triphosphates are abbreviated "dNTP.alpha.S," "dATP.alpha.S," "dCTP.alpha.S," etc.
The structure of amplification primers adapted for use in amplification reactions other than SDA is also known in the art. For example, as PCR does not require any specialized structure or sequence to sustain amplification, the PCR amplification primer typically contains only target binding sequences. However, the amplification primers of 3SR and NASBA contain an RNA polymerase promoter sequence in addition to the target binding sequences because a transcription step is required to sustain these amplification reactions.
A "bumper" or external primer is a primer which anneals to a target sequence upstream of (i.e., 5' to) an amplification primer, such that extension of the external primer displaces the downstream primer and its extension product, i.e., a copy of the target sequence comprising the restriction endonuclease recognition site is displaced. The bumper primers therefore consist only of target binding sequences and are designed so that they anneal upstream of the amplification primers and displace them when extended. External primers are designated B.sub.1 and B.sub.2 by Walker, et al. (1992b), supra. Extension of external primers is one method for displacing the extension products of amplification primers, but heating may also be suitable in certain cases.
A reverse transcription primer also consists only of target binding sequences. It is hybridized at the 3' end of an RNA target sequence to prime reverse transcription of the target. Extension of the reverse transcription primer produces a heteroduplex comprising the RNA target and the cDNA copy of the RNA target produced by reverse transcription. The cDNA is separated from the RNA strand (e.g., by heating, RNase H, or strand displacement) to make it single stranded and available for amplification. Optionally, a second reverse transcription primer may be hybridized at the 3' end of the target sequence in the cDNA to prime second strand synthesis prior to amplification.
The terms target or target sequence refer to nucleic acid sequences (DNA and/or RNA) to be amplified, replicated or detected. These include the original nucleic acid sequence to be amplified and its complementary second strand as well as either strand of a copy of the original target sequence produced by amplification or replication of the target sequence.
Amplification products, extension products or amplicons are oligonucleotides or polynucleotides which comprise copies of the target sequence produced during amplification or replication of the target sequence.
The term thermostable or thermophilic with reference to DNA-dependent DNA polymerases and other enzymes indicates that the enzymatic activity of the DNA polymerase or other enzyme is stable within the temperature range of tSDA, typically about 45.degree. C.-60.degree. C.